CN105749268A - Inactivated Zika virus vaccine - Google Patents
Inactivated Zika virus vaccine Download PDFInfo
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- CN105749268A CN105749268A CN201610222124.3A CN201610222124A CN105749268A CN 105749268 A CN105749268 A CN 105749268A CN 201610222124 A CN201610222124 A CN 201610222124A CN 105749268 A CN105749268 A CN 105749268A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides an inactivated Zika virus vaccine. The inactivated Zika virus vaccine is obtained by: performing ultrafiltration and concentration on Zika virus liquid after inactivation, centrifuging the concentrated virus liquid by adopting a sucrose density zone, performing ion exchange and concentration sterilization on a centrifugal product to obtain a Zika virus vaccine stock solution, diluting the vaccine stock solution until the total protein content is not more than 20mu g/ml, and adding an adjuvant to obtain a vaccine semi-finished product. The method for preparing the vaccine provided by the invention is simple, convenient and easy to operate, the cost is saved, the produced vaccine is suitable for Asian people, a unit dose of the Zika virus liquid is high in immunogenicity, the content of hybrid protein is low, the side effect after injection is small, and the safety is high, so that the vaccine is suitable for vaccination of fertile women before pregnancy, can avoid newborn Brazil microcephaly caused by infection of Zika virus, and is significant in social value and market efficiency.
Description
Technical field
The present invention relates to field of biological pharmacy, in particular it relates to the zika virus vaccine of a kind of inactivation.
Background technology
Zika virus sick (ZikaVirusDisease) is a kind of self limiting acute infectious disease being caused by zika virus (ZikaVirus) and being propagated by mosquito matchmaker.Zika virus was found first in nineteen forty-seven in Uganda's rhesus monkeys, and nineteen fifty-two is separated in the human body of Uganda and Tanzania.Before 2007,14 example zika virus disease Sporadic cases are only reported in the whole world, within 2007, find zika virus epidemic outbreaks on the Ya Pu island of the pacific island state Micronesia first, be then discovered that there is increase trend in the country of zika virus cases of infection and epidemic outbreaks and area.In May, 2015, the first zika virus disease case of Brazil's report, by by the end of January, 2016, Brazil waits 24 American States and area to be all found that autochthonous infection case.Meanwhile, multiple countries on the ground such as Europe, North America find introduced cases, and 1 example introduced cases from Thailand are also reported in Taiwan.
Zika virus belongs to flaviviridae (Flaviviridae) Flavivirus (Flavivirus), and spherical in shape, diameter is about 40-70nm, has peplos.Genome is single-stranded positive RNA, and length is about 10.8Kb, is divided into Asian type and Africa two genotype of type, is Asian type in the virus that South American region is popular at present.
The main manifestations infecting zika virus is heating (mostly being middle low-grade fevers), erythra (mostly being maculopapule), and can with apyetous conjunctivitis, muscle and arthralgia, malaise and headache, small number of patients may occur in which stomachache, feels sick, diarrhoea, mucosal ulcer, skin pruritus etc..The artificial inapparent infection of about 80%, there is above-mentioned clinical symptoms in the people only having 20%, and general persistently spontaneous recovery after 2-7 days, serious symptom and death are rare.There is the report infecting relevant Guillain Barre syndrome (Guillain-Barre&1& syndrome, Guillain-BarreSyndrome) case to zika virus, but cause effect relation therebetween is not yet clear and definite.Children's's cases of infection it may also occur that nervous system, eye and audition etc. change.Infection of pregnant women zika virus may result in the even foetal death of neonate microcephalus.In zika virus epidemic situation, Brazil etc. state's neonate microcephalus case load dramatically increase, epidemic situation quickly spread and and microcephalus between cause effect relation, cause the extensive concern of international community.
There is the medium yellow-fever mosquito that can propagate zika virus in south China some areas, the dengue fever Introduced cases epidemic situation that circulation way is similar with it in recent years continues to increase, and causes fairly large epidemic outbreaks in part province, south.Day by day close along with concerned countries or area people-to-people contacts, there is zika virus input risk in China.Particularly south China area summer and autumn aedes density is higher, once there be case to input, however not excluded that the possibility of local transmission diffusion occurs in some areas.
There is no effective treatment means in current global range zika virus disease is treated, even if by preventing mosquito from preventing zika virus to infect, implementation process there is also extreme difficulty, arrival especially as summer, mosquito density increases, mosquito bite cannot be taken precautions against completely at all, therefore, be undertaken preventing to become the following effective means eliminating zika virus by vaccine.
On March 1st, 2016, WHO issued stockaded village's card risk circular, and the risk in zika virus propagation and potential complication provides provisional guide.Although nineteen fifty-two has been found that zika virus first in the mankind, but is seldom related to the record of epidemic situation.From zika virus over 2015 mainly at America epidemic outbreaks, propagate aggravation, and rising also occurs in the neurological complication ratio such as microcephaly during this and Guillain-Barre&1& syndrome.From the tens of example microcephaly neonate of appearance Brazilian over 2015, in order to reduce the expansion of epidemic situation and the harm to crowd as far as possible, WHO proposes the research and development should strengthened zika virus vaccine.But there is no stockaded village's card vaccine so far and come out, immunogenicity and the stability of vaccine require study.
Owing to zika virus breaks out in this epidemic situation, although just isolated zika virus from the fifties in last century, but rarely had document to describe zika virus, its culture process, purifying process and preparation process are still blank.
Summary of the invention
In order to farthest prevention zika virus infects, better realize the target of prenatal and postnatal care simultaneously, it is an object of the invention to provide the zika virus vaccine of a kind of inactivation, it is adaptable to each age level crowd inoculates, particularly with the women of child-bearing age, effectively to take precautions against the invasion and attack of zika virus.
Another object of the present invention is to provide a kind of method preparing inactivation zika virus vaccine.
In the zika virus vaccine of inactivation provided by the invention, zika virus antigenic content is 100-1600U/ml, and total protein content is lower than 20 μ g/ml.
The preferably present invention, in zika virus vaccine, zika virus antigenic content is 800U/ml.
The zika virus vaccine of the inactivation of the present invention is prepared by following steps:
(1) it is utilized as carrier and cultivates cell technology Cultivation of Vero, when Growth of Cells becomes fine and close monolayer, inoculation zika virus work seed, cultivate,
(2) virus liquid, inactivation will be gathered in the crops after culture fluid clarification filtration；
(3) virus liquid inactivated is concentrated by ultrafiltration, and the virus liquid after concentration adopts sucrose density band centrifugation；
(4) centrifugal product ultrafiltration desaccharide, obtains desaccharide liquid；Desaccharide liquid is carried out ion exchange, obtains chromatographic solution；Chromatographic solution carries out concentrating, degerming be stock solution；
(5) stock solution is diluted, add adjuvant, obtain vaccine semi-finished product.
Zika virus culture medium provided by the invention is that M199 and DMEM/F12 mixes according to 1:1 equal-volume.
On the other hand, a kind of method that the invention provides zika virus vaccine preparing inactivation, comprise the following steps:
(1) it is utilized as carrier and cultivates cell technology Cultivation of Vero, when Growth of Cells becomes fine and close monolayer, inoculation zika virus work seed, cultivate；
(2) virus liquid, inactivation will be gathered in the crops after culture fluid clarification filtration；
(3) virus liquid inactivated is concentrated by ultrafiltration, and the virus liquid after concentration adopts sucrose density band centrifugation；
(4) centrifugal product ultrafiltration desaccharide, obtains desaccharide liquid；Desaccharide liquid is carried out ion exchange, obtains chromatographic solution；Chromatographic solution carries out concentrating, degerming be stock solution；
(5) stock solution is diluted, add adjuvant, obtain vaccine semi-finished product.
Wherein, step (1), when Growth of Cells becomes fine and close monolayer, is that 0.05-0.3 inoculates zika virus work seed according to MOI, and cultivation temperature is 35-37 DEG C, incubation time is 72-96 hour, and the culture medium of virus is that M199 and DMEM/F12 mixes according to 1:1 equal-volume.
In the inventive method, in step (2), ablation method is the formaldehyde adding final concentration of 200 μ g/ml in single harvest liquid, 37 DEG C, inactivates 4-7 days.
Wherein, step (3) virus liquid cycles of concentration is 10-100 times, and film bag can trapped molecular weight be 100-300KD.
Further, the sucrose density band centrifugation method of step (3) is: surpass from buffer from lateral opening pump into successively with 50-100rpm of centrifuge successively, virus liquid after ultrafiltration concentration, low concentration sucrose solution, finally pump into high concentration sucrose solution with 50-150rpm until mesopore trickle centrifugal rotational speed is 28000-35000rpm, centrifugation time is: 6-20 hour, and centrifuging temperature is 2-8 DEG C；
Preferably, centrifugal rotational speed is 30000rpm, and centrifugation time is: 14 hours, and centrifuging temperature is 4 DEG C.
Low concentration sucrose solution sucrose mass percent is 25%-45%, and high concentration sucrose solution sucrose mass percent is 55%-60%, and surpassing from buffer is 0.01-0.05MPBS, and pH value is: 6.0-7.5.
In the inventive method, step (5) adjuvant is aluminum hydroxide adjuvant, is 1:1 with the stock solution volume after dilution
In the inventive method, step (5) is that stock solution is diluted to 200-3200U/ml, according to 1:1 volume ratio and aluminium adjuvant mixing.
The present invention possesses following beneficial effect:
(1) present invention prepares the vaccine effectively protecting zika virus first; can largely alleviate this zika virus epidemic situation; it is significantly reduced sufferer; good guarantee is provided for prenatal and postnatal care; the women of child-bearing age inoculate this vaccine before pregnant can protect the invasion and attack of pregnancy period zika virus; avoid the generation of the similar newborn child's illnesss such as Brazil's microcephaly, thus alleviating the burden of society and family.The strain that the present invention adopts is that domestic Introduced cases zika virus blood samples of patients separates, and is applicable to Asia ethnic group immunity with virus for the vaccine that antigen prepares.The present invention is domestic large-scale culture zika virus first, and prepares zika virus vaccine.
(2) cell that the present invention uses is Vero cell, and this cell has growth rapidly, it is possible to form the advantage of fine and close monolayer, and its safety obtains international extensively certification.Use Vero cell can realize the purpose of Multiple harvests, operating procedure can be significantly reduced and reduce cost.What the present invention adopted is microcarrier Cultivation of Vero, screening through research worker, prepare the culture medium that applicable zika virus produces, thus turning out high order of magnitude high density to be more simply and rapidly suitable to the cell of virus inoculation, save production cost, improving production efficiency, the industrialization being suitable for product produces.
null(3) purification schemes of virus liquid is by the present invention: carry out EXPERIMENTAL DESIGN by DOE，Zika virus best medium is drawn by lot of experimental data，Virus liquid is concentrated by ultrafiltration、Density gradient centrifugation、The purification process steps such as ion exchange are simple can utilize simple and efficient being purified into of advantage of density gradient to have immunogenic intact virus simultaneously，Effectively will containing E protein、The complete zika virus granule of M albumen and C protein，The method relatively chromatography method i.e. simplicity but also economic，Not only increase the immunogenicity of unit dose zika virus liquid and reduce the side reaction after the vaccine prepared with this virus liquid uses，Reduce production cost simultaneously，And the operating process of sucrose density gradient centrifugation simplicity and low production cost have commercial viability，Compare chromatography method simultaneously and can remove impurity protein preferably，Ensure that the safety of vaccine.
Accompanying drawing explanation
Fig. 1 is the SDSPAGE electrophoresis result figure after embodiment 3 virus liquid density gradient centrifugation.1-9 swimming lane represents the 10th, 11,12,13,14,15,16,17, the 18 pipe centrifugal liquid that density gradient centrifugation is collected respectively.Wherein 10-16 pipe is mainly impurity protein, and including Ox blood serum residual protein, host cell proteins, and 17-18 pipe is target viral, includes 3 result albumen E, M, C protein of zika virus.
Fig. 2 is zika virus stock solution HPLC testing result.
Detailed description of the invention
Following example further illustrate present disclosure, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the amendment that the inventive method, step or condition are made or replacement, belong to the scope of the present invention.
If not specializing, the conventional means that technological means used in embodiment is well known to those skilled in the art；Reagent used in embodiment is commercial goods.
Zika virus of the present invention is from Zhejiang Center For Disease Control and Prevention.
The preparation of embodiment 1 cell work seed and the preparation of virus liquid
1, high-density cells work Spawn preparation
Using 199 culture fluid dilution Vero chief cell seed dilution ratios is 60:1, is inoculated into 175cm2Cultivating in cell square vase, cultivation temperature is 36.0 DEG C ± 1 DEG C.Treat that cell is paved with cell bottle, continue, by 1:4～1:6,4 generations of going down to posterity.In last generation, reaches 5 40 layer cell factory, carries out trypsinization, is centrifuged and removes supernatant.Using the frozen stock solution containing 10%DMSO to carry out resuspended, after mixing, cell concentration is 2.5 × 107Individual/ml.Cell seed is carried out gradient cooling at programmed cooling instrument, keeps decline per minute 1 DEG C.Temperature moves to after being down to-196 DEG C in liquid nitrogen and preserves, and the effect duration of working cell seed is 10 years.
2, Vero cell is cultivated
(1) one-level cell is cultivated and is weighed microcarrier, swelling overnight with 0.01MPBS, rinses 2 times with appropriate PBS, is placed in the 121 DEG C of sterilizings of high pressure steam sterilization cabinet.Being taken out from liquid nitrogen by cell seed, shake rapidly thawing in the water-bath of 39 ± 1 DEG C, after counting, add in growth-promoting media, carry out one-level cell cultivation, volume of culture is about 5L.Adjusting each culture parameters, temperature is 36.5 DEG C, pH value is 7.20, dissolved oxygen is 50%, rotating speed is 30rpm.Cell is cultured to during plateau 2.5 × 106Cells/ml, carries out had digestive transfer culture.
(2) secondary cell is cultivated and through digestion, the cell that step (1) has been cultivated is prepared into cell suspension, it is seeded to 20L cell reactor, adjusting each culture parameters, temperature is 36.5 DEG C, pH value is 7.20, dissolved oxygen is 50%, rotating speed is 30rpm, starts to cultivate.Cell is cultured to during plateau 5 × 106Cells/ml, is ready for digestion.
(3) three grades of cell culture step (2) cultivated after cell prepare into cell suspension, be seeded in working volume 120L cell reactor, arrange temperature be 36.5 DEG C, pH value be 7.20, rotating speed be 30rpm, dissolved oxygen be 50%.Cell density is cultured to 1.5 × 106Cells/ml prepares virus inoculation.
3, virus liquid is cultivated and is pressed the work seed inoculation of MOI0.05 zika virus, culture parameters is adjusted to rotating speed: 30rpm, temperature: 36.5 DEG C, pH value: 7.30, dissolved oxygen: 50%.Cultivate 3 days, harvesting supernatant, be zika virus harvest liquid.
The screening of embodiment 2 culture medium
Select MEM, M199, DMEM, the DMEM/F12 culture medium that market is sold, adopt the DOE software of Minitab17 to carry out EXPERIMENTAL DESIGN, carry out Virus culture according to the culture medium prescription designed, compare virus titer, viral immunogenic result, select best medium.The culture medium prescription of EXPERIMENTAL DESIGN is as shown in table 1, and wherein " 1 " represents and adds this culture medium, and "-1 " represents without this culture medium.Formula there is several " 1 " just carry out a few decile, each culture medium respectively decile.Virus titer and immunogenicity result such as table 2, shown in table 3, viral immunogenic and the titre of the wherein culture medium production that M199 and DMEM/F12 mixes according to 1:1 equal-volume are significantly higher than other culture medium.
Table 1DOE test medium formula
Table 2 virus titer result
| Formula | Titre results (CCID50/ml) |
| ① | 7.0 |
| ② | 6.5 |
| ③ | 6.9 |
| ④ | 7.3 |
| ⑤ | 7.1 |
| ⑥ | 6.8 |
| ⑦ | 7.9 |
Table 3 immunogenicity result
| Formula | GMT (1 :) |
| ① | 512 |
| ② | 384 |
| ③ | 512 |
| ④ | 768 |
| ⑤ | 768 |
| ⑥ | 512 |
| ⑦ | 1024 |
7. number culture medium is selected, as zika virus culture medium according to immunogenicity and titre results.
The inactivation of embodiment 3 zika virus and purification
1, zika virus inactivation zika virus harvest liquid adds final concentration of 200 μ g/ml formaldehyde, put 37 DEG C and inactivate 7 days.Namely the inactivation of zika virus is completed.
2, zika virus purification
(1) clarification by virus liquid through filter element filtering that continuous 3 grades of apertures are 3-0.8 μm, 0.8-0.65 μm, 0.65-0.22 μm or centrifugal (including continuous flow centrifugation) rotating speed be 2000-5000rpm, centrifugation time is 0.5-4 hour, obtains virus clear liquor；
(2) concentrating virus clear liquor concentrates through the Pall slipstream membrane filtration system of 300KD molecular cut off, and cycles of concentration is 100 times.
(3) low, the high concentration of density gradient centrifugation sucrose solution respectively 25%, 55%.Density gradient centrifugation buffer is 0.05mol/LPBS buffer, and its pH value is: 6.5.Use Hitachi's CP70MX/ME supercentrifuge, 100ml ultracentrifugation buffer is pumped into 100rpm successively from lateral opening, the embodiment 1 of about 700ml is concentrated by ultrafiltration the zika virus ultrafiltrate obtained, low concentration sucrose solution 500ml, finally pump into high concentration sucrose solution with 100rpm until mesopore trickle, be centrifuged 10h at 4 DEG C through 30000rpm ultrafiltration.After being centrifuged, 500ml collected by the first pipe, collects with 50ml centrifuge tube afterwards, and 50ml/ manages, it is possible to collect 25-27 pipe.Detect antigenic content and the protein content of often pipe, merged the centrifugal liquid containing E, M, C protein by protein electrophoresis result.The SDSPAGE electrophoresis result of each pipe is shown in Fig. 1, it was demonstrated that density gradient centrifugation can by containing E, M, C protein zika virus efficiently separate.
(4) the density gradient centrifugation liquid in desaccharide step (3) carries out desaccharide through the Pall slipstream membrane filtration system of 100KD molecular cut off.
(5) ion exchange ion displacement chromatography medium is DEAEFF, and level pad is 0.02MPBS solution, and elution buffer is 0.02MPBS, loading flow velocity and clean flow velocity respectively 15ml/min, 80ml/min.Collecting when stream wears peak and flex point occur downwards for boundary with antigen peak, front and back are divided into two sections of collections, are chromatographic solution.
(6) chromatographic solution in purified concentration step (5) concentrates through the Pall slipstream membrane filtration system of 100KD molecular cut off, filters through 0.22um and be stock solution after concentrating 100 times.Stock solution detects purity as in figure 2 it is shown, purity reaches 98.6% by HPLC.
Embodiment 4 zika virus stock solution prepares into the indices evaluation of vaccine
1, this step product of impurity content is zika virus stock solution
Zika virus stock solution embodiment 2 obtained is diluted to 600U/ml according to antigenic content, and final half-finished antigenic content is 800U/ml, and total protein content is lower than 20 μ g/ml.It is subsequently added；Aluminium adjuvant standby one-tenth zika virus vaccine finished product.The impurity content result of vaccine finished product is in Table 4.
46 crowdes of zika virus vaccine finished product major impurity residual quantity result * of table
If * calculating numerical value during P value is less than (<) value is calculated according to this value.If numerical value is value range, it is calculated according to the meansigma methods of scope bound.
2, immunogenicity
3 times of serial dilutions are carried out, totally 4 dilution factors after being melted again by vaccine finished product；Each dilution factor sample carries out single two hindlimb muscle immune rat respectively, and only, 10 (male and female half and half) are dilution factor often for 0.5ml/, immunity blood sampling in latter 21 days, separating serum, the NAT of anti-zika virus in detection serum, calculates serological conversion rate respectively.Serological conversion rate and neutralizing antibody geometrical mean GMT level are in Table 5.
Table 5 stockaded village card vaccine finished product immunogenicity result
| Batch | 1 | 2 | 3 | 4 | 5 | 6 |
| Former times of Conversion rate | 100% | 100% | 100% | 100% | 100% | 100% |
| GMT (1 :) | 768 | 2048 | 1024 | 1536 | 1024 | 768 |
3, stability
Vaccine finished product is stored under 37 DEG C of environment, temporally sampling detection antigenic content, outward appearance, pH value, osmotic pressure molar density, bacterial endotoxin, BSA content, HCP content, DNA content etc..Record data to 8 weeks, result table 6-11.
637 DEG C of finished products of table-antigenic content testing result (percentage ratio of labelled amount)
* the percent value of labelled amount is the percent value of relatively 0 month value.
737 DEG C of DNA content testing results of table
837 DEG C of BSA content detection results of table
937 DEG C of HCP content detection results of table
1037 DEG C of osmotic pressure molar density testing results of table
1137 DEG C of detection of bacterial endotoxin results of table
Although, above the present invention is described in detail with a general description of the specific embodiments, but on basis of the present invention, it is possible to it is made some modifications or improvements, and this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.
Claims (10)
1. the zika virus vaccine of an inactivation, it is characterised in that zika virus antigenic content is 100-1600U/ml, and total protein content is lower than 20 μ g/ml.
2. the zika virus vaccine of inactivation as claimed in claim 1, it is characterised in that zika virus antigenic content is 800U/ml.
3. the zika virus vaccine of inactivation as claimed in claim 1, it is characterised in that prepared by following steps:
(1) it is utilized as carrier and cultivates cell technology Cultivation of Vero, when Growth of Cells becomes fine and close monolayer, inoculation zika virus work seed, cultivate；
(2) virus liquid, inactivation will be gathered in the crops after virus-culturing fluid clarification filtration；
(3) virus liquid inactivated is concentrated by ultrafiltration, and the virus liquid after concentration adopts sucrose density band centrifugation；
(4) centrifugal product ultrafiltration desaccharide, obtains desaccharide liquid；Desaccharide liquid is carried out ion exchange, obtains chromatographic solution；Chromatographic solution carries out concentrating, degerming be stock solution；
(5) stock solution is diluted, add adjuvant, obtain vaccine semi-finished product.
4. vaccine as claimed in claim 3, it is characterised in that the virus-culturing fluid described in step (2) is that M199 and DMEM/F12 is mixed to get according to volume ratio 1:1.
5. the method for the zika virus vaccine preparing inactivation, it is characterised in that comprise the following steps:
(1) it is utilized as carrier and cultivates cell technology Cultivation of Vero, when Growth of Cells becomes fine and close monolayer, inoculation zika virus work seed, cultivate；
(2) virus liquid, inactivation will be gathered in the crops after virus-culturing fluid clarification filtration；
(3) virus liquid inactivated is concentrated by ultrafiltration, and the virus liquid after concentration adopts sucrose density band centrifugation；
(4) centrifugal product ultrafiltration desaccharide, obtains desaccharide liquid；Desaccharide liquid is carried out ion exchange, obtains chromatographic solution；Chromatographic solution carries out concentrating, degerming be stock solution；
(5) stock solution is diluted, add adjuvant, obtain vaccine semi-finished product.
6. method as claimed in claim 4, it is characterised in that step (1), when Growth of Cells becomes fine and close monolayer, is that 0.05-0.3 inoculates zika virus work seed according to MOI, and cultivation temperature is 35-37 DEG C, and incubation time is 72-96 hour.
7. method as claimed in claim 4, it is characterised in that in step (2), ablation method is the formaldehyde adding final concentration of 100-400 μ g/ml in single harvest liquid, 37 DEG C, inactivates 4-7 days.
8. method as claimed in claim 4, it is characterised in that step (3) virus liquid cycles of concentration is 10-100 times, and film bag can trapped molecular weight be 100-300KD.
9. the method as described in as arbitrary in claim 4-7, it is characterized in that, the sucrose density band centrifugation method of step (3) is: surpass from buffer from lateral opening pump into successively with 50-100rpm of centrifuge successively, virus liquid after ultrafiltration concentration, low concentration sucrose solution, finally pumping into high concentration sucrose solution with 50-150rpm until mesopore trickle centrifugal rotational speed is 28000-35000rpm, centrifugation time is: 6-20 hour, and centrifuging temperature is 2-8 DEG C；
Low concentration sucrose solution sucrose mass percent is 25%-45%, and high concentration sucrose solution sucrose mass percent is 55%-60%, and surpassing from buffer is 0.01-0.05MPBS, and pH value is: 6.0-7.5.
10. the method as described in as arbitrary in claim 4-7, it is characterised in that stock solution is diluted to 200-3200U/ml, mixes according to 1:1 equal-volume with adjuvant.
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